Decrease in beech ( Fagus sylvatica ...

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The aim of this study was to de ter mine if the loss of germi - nability and vi a bil ity of beech (Fagus sylvatica L.) seeds stored at dif fer ent vari ants of tem per a ...
ACTA PHYSIOLOGIAE PLANTARUM Vol. 27. No. 1. 2005: 3-12

Decrease in beech (Fagus sylvatica) seed viability caused by temperature and humidity conditions as related to membrane damage and lipid composition Ewelina Ratajczak, Stanisława Pukacka* Institute of Dendrology, Polish Academy of Sciences, ul. Parkowa 5, 62-035 Kórnik * corresponding author, e-mail: [email protected]

Key words: fatty acids, lipid peroxidation, membrane permeability, phospholipids, sterols, α-tocopherol, viability

Abstract The aim of this study was to determine if the loss of germinability and viability of beech (Fagus sylvatica L.) seeds stored at different variants of temperature (4, 20, and 30 °C) and relative humidity (RH: 45 and 75 %) is associated with a loss of membrane integrity and changes in lipid composition. Beech seeds stored for 9 weeks gradually lost viability at a rate dependent on temperature and humidity. The harmful effect of temperature increased with growing humidity. The loss of seed viability was strongly correlated with an increase in membrane permeability and with production of lipid hydroxyperoxides (LHPO), which was regarded as an indicator of peroxidation of unsaturated fatty acids. The condition of membranes was assessed on the basis of their permeability and the state of lipid components: phospholipids and fatty acids. During seed storage we observed a decline in concentration of individual phospholipids and fatty acids, proportional to the loss of seeds viability. We also detected a decrease in concentrations of α-tocopherol and sterols, which play an important role in protection of membranes against the harmful influence of the environment. Our results show that the germinability of beech seeds declines rapidly at temperature above 0 °C and growing humidity. This is due mainly to the loss of membrane integrity, caused by peroxidation of unsaturated fatty acids.

Introduction

Loss

of viability with seed ageing is connected mainly with loss of membrane integrity (Priestley 1986, Pukacka 1991, Bewley and Black 1994). Recent research conducted by Wolkers and Hoekstra (2003) with the use of a spectrometric method (FTIR) has confirmed that in ageing seeds the secondary structure of proteins remains unchanged, while marked changes are observed in membrane structure. The major cause of the loss of membrane integrity is believed to be the peroxidation of unsaturated fatty acids, which may take place with participation of ROS or the enzyme lipoxygenase (Wilson and Mc Donald 1986, Mc Donald 1999). As a consequence of the process initiated by free radicals, such as the hydroxyl radical (OH.), reactive forms of fatty acids (ROO.) are generated, but the mechanism of this reaction has not been fully explained yet (Halliwell and Gutteridge 1989). Apart from changes in unsaturated fatty acids, free radicals may also cause de-esterification of phospholipids and accumulation of free fatty acids (FFA) and lysophospholipids (LPLs) (Mc Kersie et al. 1988, Van Bilsen et al. 1994). Those unfavourable reactions initiate changes in membranes, such as a loss of their fluidity, the increase in the gel-to-liquid crystalline phase transition temperature

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E. RATAJCZAK & S. PUKACKA

(Tm). All those changes lead to increased permeability of membranes and cell death. Lipid peroxidation is usually detected by means of assaying its products, such as volatile aldehydes or malondialdehyde (Esashi et al. 1997, Sung and Jeng 1994). Also analysis of changes in lipid components of membranes - like phospholipids and their fatty acids - enables determination of the causes of changes in membrane integrity (Pukacka 1991, Pearce and Samad 1980). However, in some studies no changes in fatty acids were detected, especially in seeds subjected to accelerated ageing (Priestley and Leopold 1979, Corbineau et al. 2002).

One of the most effective factors protecting membranes against the harmful effects of free radicals is a-tocopherol. This low-molecular antioxidant is located directly inside the lipid layer of membranes and effectively inactivates free oxygen radicals and slows down the process of production of organic radicals. A decrease in concentration of α-tocopherol in the course of seed ageing, was detected in soybean by Senaratna et al. (1988) and in Norway maple by Pukacka (1991). Zalewski et al. (2000) showed that a-tocopherol-treated seeds of the field bean (Vicia faba var. minor) were characterized by a much higher viability and vigour after storage for several years, as compared to non-treated seeds. However, Merritt et al. (2003) did not detect any correlation between seed viability during storage and α-tocopherol content of seeds of several Australian plant species.

The aim of this study was to assess changes in lipid components of cell membranes in beech seeds during storage in various temperature and humidity conditions in relation to membrane permeability and seed viability. Seeds of this species are assigned to the suborthodox category and their germinability declines quickly in unfavourable storage conditions.

Material and methods Plant material and storage conditions

Beech seeds were collected in the autumn of 2000 in the Krucz Forest District (NW Poland). After desiccation to 9 % moisture content (MC), they were stored at –10 °C in a seed-bank in Białogard.

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From there, they were transported to our laboratory in plastic bags in a cooling box and next stored in laminated aluminium foil bags at –10 °C. The germinability of these seeds reached 93 %.

To investigate the effect of temperature and humidity, aliquots of seed samples were stored for 1 week at two humidity levels: 45 and 75 % RH (established above respective saturated salt solutions of K2CO3 and NaCl) (Scandé et al. 2000), and at three temperatures: 4, 20 and 30 °C. After that time, the seeds were placed in laminated aluminium foil bags, hermetically sealed, and stored for additional 8 weeks at respective temperatures. After 3, 6 and 9 weeks of storage, seeds were used for analyses. Non-treated seeds stored at –10 °C were the control. Germination test

Samples of 2×100 seeds from each variant were allowed to germinate at 3 °C, after careful imbibition, between moist rolled paper towels in separate boxes. Normal germination was assessed according to ISTA (1999). Electrolyte leakage

Three samples of 20 seeds each, deprived of pericarp and seed coat were placed in 10 ml of deionized water. Conductivity of the solutions was measured after 24 hours of incubation at room temperature. Results were expressed in mS⋅g-1DW. Analysis of lipid hydroxyperoxides (LHPOs)

LHPOs were determined according to the method of Griffiths et al. (2000). Three samples of 5 embryos (ca 1 g) of each treatment were taken to lipid extraction using chloroform:methanol (1:2, v/v) solvent. All procedures were performed in dim light in a cold room, using chilled solvents and amber vials. Absorbances were determined spectrophotometrically at 560 nm and the concentration of LPHOs determined with a molar absorption coefficient derived for standard linoleate hydroperoxide (ε=6.0×104 M-1⋅cm-1, Gay et al. 1999).

VIABILITY OF FAGUS SYLVATICA SEEDS AS RELATED TO MEMBRANE DAMAGE

Lipid extraction and analyses of phospholipids (PL), fatty acids (FA), α-tocopherol and sterols

Three samples of 50 embryo axes each and three samples of 20 cotyledons each were subjected to lipid extraction using chloroform: methanol (2:1, v/v) supplemented with 0.05 % buthylated hydroxytoluene (BHT) according to Allen et al. (1966), as described by Pukacka (1983). Aliquots of lipid extracts were separated on Sep-Pak silica cartridge (Waters Associates) according to Juaneda and Rocquelin (1985) and phospholipid fractions were obtained.

Particular

phospholipids were de ter mined in aliquots of phospholipid fractions by separation on silica gel TLC plates (Merck) in a solvent chloroform : methanol: acetic acid: water (85:15:10:3.5, v/v/v/v) (Nichols et al. 1965), with the use of original phospholipid standards. Spots con taining phospholipids were detected with iodine vapour and were scraped off for phosphorus analysis. The phosphorus content in spots was estimated according to Ames (1966). Fatty acids were determined in aliquots of phospholipid fractions. Samples with heptadecanoic acid (17:0) as the internal standard were saponified and methylated according to Metcalfe et al. (1966). They were separated by using a Hewlett Packard gas chromatograph equipped with Supelco 2330 capillary column (30 m×0.25 mm), at 220 °C (in isotherm), with He as the car rier g as. Free f atty ac ids (F FA), α-tocopherol, and sterols were determined in aliquots of the total lipid fractions according to Kendal and McKersie (1989) after silylation of samples with B S T FA ( N - O - b i s-trimethylsilyl-trifluoro-acetamide) and pyridine at room temperature in the darkness. Silylated samples and internal standards (heptadecanoic acid 17:0, and α-cholestan) were separated on the gas chromatograph with a SPB 1 (Supelco) microcapillary column and identified by comparing retention times with those of authentic standards. Quantities of FFA were the sum of myristic + palmitic + stearic + oleic + linoleic + linolenic acids in total lipid extract. Concentrations of sterols and α-tocopherol were expressed in mg⋅g-1DW.

Statistical analysis

Data are presented as means ± standard deviation of six replicates. The statistical differences between seeds stored at different temperature and humidity conditions and the control (non-treated) were tested using an analysis of variance (ANOVA). Levels of significance are indicated as * P